Method for diagnosis of pancreatic exocrine function

ABSTRACT

A preparation for the diagnosis of pancreatic exocrine function by determining the amount in which a substance administered to a subject or a degradation product or metabolite thereof migrates into the blood and/or is excreted out of the body, wherein the substance is carried by a carrier and released from the carrier when exposed to the action of a pancreatic exocrine function-related factor.

This is a continuation of application Ser. No. 10/362,639, filed Feb.25, 2003, now U.S. Pat. No. 7,125,683, which claims the benefit ofPCT/JP01/07528 filed Aug. 31, 2001, which in turn claims the benefit ofJP2000-272116 filed Sep. 7, 2000, all of which are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to preparations for the diagnosis ofpancreatic exocrine function, drug delivery systems for a medical agentand enteric preparations.

BACKGROUND OF THE INVENTION

“Pancreatic exocrine function tests” are useful for the diagnosis ofpancreatic diseases such as chronic and acute pancreatitis andpancreatic cancer. They are also useful for the ascertainment of thecondition of a disease, the management of medication and the assessmentof the prognosis of a disease in a patient. (As for the general review,see Arvanitakis and Cooke, Gastroenterology, 74:932 (1978); Niederau andGrendell, Gastroenterology, 88:1973 (1985); Goldberg, Bull. Mol. Biol.Med., 15:1 (1990); Lankisch, Int. J. Pancreatology, 14:9 (1993); Bankand Chow, Gastroenterologist, 2:224 (1994); and Steer et al., New Eng.J. Med., 332:1482 (1995).

The standard method for pancreatic exocrine function tests is one whichinvolves inserting a tube through the mouth to the duodenum to collectthe duodenal juice. At present, the secretin test is commonly usedwherein secretin is intravenously administered to stimulate thesecretion of the pancreatic juice prior to the collection. This methodis highly accurate since the amounts and components of the pancreaticjuice are directly analyzed. However, this method can not be usedrepeatedly or used for screening because of the very strong stresscaused on the patients. It is only available in a relatively smallnumber of medical facilities staffed with highly skilled physiciansFurther, since this method requires fluoroscopic tube placement duringthe collection of the duodenal juice, there is a problem of X rayexposure.

Hence, in repeated or screening tests, simpler methods have been usedwhich require no intubation. At present, the following four methods aremainly used:

1. PED test wherein a synthetic substrate BT-PABA(N-benzoyl-L-tyrosyl-p-aminobenzoic acid) for chymotrypsin secreted fromthe pancreas is orally administered and the urinary excretion of PABA(p-aminobenzoic acid), the product of degradation by chymotrypsin, isdetermined;

2. PLT test wherein a synthetic substrate FDL (fluorescein dilaurate)for cholesterol ester hydrolase, esterase, secreted from the pancreas isorally administered and the urinary excretion or the blood level of thedegradation product fluorescein is determined;

3. Fecal chymotrypsin test wherein chymotrypsin in the feces isquantitatively determined; and

4. Fecal elastase test wherein elastase in the feces is quantitativelydetermined.

However, the sensitivity of any of these tests is too low to detectslight decreases in pancreatic exocrine function.

Furthermore, the PFD and PLT tests require a long time to carry out andtherefore cannot often be performed on outpatients and are not suitablein physical examinations or the like.

To solve these problems, a number of simpler pancreatic exocrinefunction tests have been reviewed. ¹³C-breath tests have also beenapplied wherein a ¹³C-labeled compound is administered and an increasein the concentration of ¹³CO₂ in the breath is determined. The followingtests may be listed:

1. ¹³C-breath test wherein a ¹³C-labeled lipid or mixed triglyceride,which is a substrate for lipase, is administered (Chen et al., J.Nuclear Med., 15:1125 (1974); Watkins et al., J. Lab. Clin. Med., 90:422(1977); Ghoos et al., Digestion, 22:239 (1981); John, S G.,Gastroenterology, 83:44 (1982); Watkins et al., Gastroenterology, 82:911(1982); Benini et al., Digestion, 29:91 (1984); Jones et al., J. Lab.Clin. Med., 105:647 (1985); Knoblach et al., Monatsschr Kinderheilkd,136:26 (1988); Vantrappen et al., Gastroenterology, 96:1126 (1989);Murphy et al., Arch. Disease in Childhood, 65:574 (1990); Kato et al.,Am. J. Gastroenterol., 88:64 (1993); McClean et al., Arch. Disease inChildhood, 69:366 (1993); Jakobs et al., Eur. J. Pediatr., 156:S78(1997); and Kalivianakis et al., Eur. J. Clin. Invest., 27:434 (1997));

2. ¹³C-breath test wherein a ¹³C-labeled cholesterol ester, which is asubstrate for cholesterol esterase, lipase, is administered (Mundlos, etal., Pediatric Res., 22:257 (1987); Cole et al., Gastroenterology,93:1372 (1987); and Mundlos et al., Gut, 31:1324 (1990));

3. ¹³C-breath test wherein a ¹³C-labeled starch, which is a substratefor amylase, is administered (Hiele et al., Gastroenterology, 96:503(1989); Dewit et al., Pediatric Res., 32:45 (1992); and Z.Gastroenterol., 35:187 (1997));

4. ¹³C-breath test wherein a ¹³C-enriched egg protein, which is aprotein having the ¹³C-concentration increased from the naturalabundance of 1.1 atm % up to 1.4 atm % by feeding a chicken with¹³C-leucine and which is a substrate for protease, is administered (Y.Ghoos, ¹³CO₂-Breath Tests at the laboratory “Digestion-Absorption”,University Hospital Gasthuisberg, Leuven, Belgium (1996)); and

5. breath test wherein a ¹³C-labeled peptide (Japanese Unexamined PatentApplication Publication No. 2000-053697), a ¹³C-labeled fluoresceinester compound (Japanese Unexamined Patent Application Publication No.2000-159773) or a ¹³C-labeled oligosaccharide or acyclodextrin/¹³C-labeled compound clathrate inclusion complex (JapaneseUnexamined Patent Application Publication No. 2000-159810) isadministered.

However, the methods 1 to 4 are also low in sensitivity andtime-consuming. Therefore, these methods have not been established inclinical fields. In the method 5, the ¹³C-labeled compounds currentlyused in the test are expensive.

Under these situations, development of a simple pancreatic exocrinefunction test that imparts low stress on a subject, is less costly andgives accurate results in a short time has been demanded.

Thus, it is an object of the present invention to provide a diagnosticpreparation for the diagnosis of pancreatic exocrine function whichallows a simple, highly sensitive pancreatic exocrine function test thatimparts low stress on a subject, is less costly and gives accurateresults in a short time.

It is another object of the present invention to provide a new drugdelivery system and enteric preparation.

DISCLOSURE OF THE INVENTION

The present inventors have found that using a preparation comprising acompound carried by a carrier composed of a substrate for a pancreaticexocrine enzyme, one can estimate pancreatic exocrine function byutilizing the phenomenon that the carrier is degraded when exposed tothe action of the pancreatic exocrine enzyme so that the release rate ofthe compound from the carrier is enhanced. Thus, the present inventionwas completed.

Thus, the present invention provides a preparation for the diagnosis ofpancreatic exocrine function by determining the amount in which asubstance administered to a subject or a degradation product ormetabolite thereof migrates into the blood and/or is excreted out of thebody, wherein the substance is carried by a carrier and released fromthe carrier when exposed to the action of a pancreatic exocrinefunction-related factor.

The present invention also provides a drug delivery system comprising amedical agent carried by a carrier, wherein the medical agent isreleased from the carrier when exposed to the action of a pancreaticprotease. In the drug delivery system, the target of the medical agentcan be the superior part of the small intestine.

The present invention further provides a preparation comprising amedical agent carried by a carrier, wherein the medical agent isreleased from the carrier when exposed to the action of a pancreaticprotease. The preparation can be used as an enteric preparation.

As used herein, the term “carrier” refers to any material that serves toretain and/or stabilize a substance administered to a subject or patient(e.g., a variety of elements and compounds) in a preparation and/orserves to deliver the substance to a desired part in the living body.Examples of the carrier include polymeric support media such assolidified egg white, lipids, polysaccharides, starch gels and albumin;particulate preparations such as tablets, capsules, powder and granules;and bio-derived substances such as erythrocytes and lipoproteins.

As used herein, the term “carry” means “to keep carrying”. The manner inwhich a substance is carried by a carrier includes the following: thesubstance is dispersed and/or encapsulated in the carrier; the substanceis adsorbed on the carrier; the substance is bound to the carrier; andso on.

As used herein, the term “disperse” or “dispersion” means that asubstance is present as fine particles in a medium that is differentfrom the substance. The fine particles can have variable particle sizes,and may be present as coarse particles (1 μm or greater), colloidalparticles (1 μm to 1 nm) or molecules (10 nm or smaller). The manner inwhich a substance is dispersed in a carrier includes the following; thesubstance is contained in a matrix formed by the carrier (e.g., apolymeric matrix); molecules or particles of the substance are presentwithin or between molecules of a material constituting the carrier;molecules or particles of the substance are present in the space(s)formed by one or more molecules of a material constituting the carrier;and so on.

As used herein, the term “encapsulate” or “encapsulation” means “tocontain a substance and enclose it”. The manner of encapsulationincludes the following: a substance is encapsulated in microcapsules; asubstance is covered with an outer coating; a protein contains a metalion; and so on.

As used herein, the term “adsorb” or “adsorption” means that a substanceis enriched or concentrated at the interface between two phases. Anexemplary manner of adsorption is such that a substance is concentratedon the surface of a matrix formed by other substance (e.g., a polymericmatrix) via an ionic bond or the like.

As used herein, the term “bind” or “binding” means a concept includingany type of binding of a substance to a carrier via a covalent bond(e.g., an ester bond). As an example, albumin-ethyl ester fine particlesmay be mentioned in which ethanol is bound to carboxyl groups in theprotein via an ester bond. The substance to be bound to albumin may beany compound as long as it can bind to a carboxyl or hydroxyl group inalbumin via an ester bond and if it is harmless and can bequantitatively determined in its free form. Examples of the compoundinclude alcohols that are non-toxic to humans, such as ethanol; organicacids such as formic acid, acetic acid, propionic acid, pyruvic acid andbenzoic acid; saccharides such as glucose; and amino acids such asalanine.

As used herein, the term a “pancreatic exocrine function-related factor”refers to a substance or environmental factor that is involved in orcontributes to the pancreatic exocrine function, including, e.g.,pancreatic exocrine enzymes (e.g., protease, lipase, amylase), pH of theintestinal juice, bicarbonate ion, sodium ion, chlorine ion, calcium ionand the amount or volume of the intestinal juice.

As used herein, the term a “medical agent” refers to any one of avariety of elements and compounds used for medicinal purposes.

As used herein, the term “enteric” refers to a property of beingdissolved/disintegrated only after reaching the intestine without beingdissolved/disintegrated in the stomach, or a property of beingdissolved/disintegrated by low degree in the stomach but beingdissolved/disintegrated by an increased degree in the intestine.

Hereinafter, the present invention will be described in detail.

The diagnostic preparation for pancreatic exocrine function of thepresent invention is a preparation for the diagnosis of pancreaticexocrine function by determining the amount in which a substanceadministered to a subject or a degradation product or metabolite thereofmigrates into the blood and/or is excreted out of the body. Thesubstance to be administered to a subject (hereinafter, also referred toas “administered substance”) is carried by a carrier, e.g., in such away that the substance is dispersed and/or encapsulated in the carrier,is adsorbed on the carrier, or is bound to the carrier. The carrierreleases the carried substance under the action of a pancreatic exocrinefunction-related factor. In one embodiment of the present invention, thecarrier experiences changes in its structure (e.g., decomposition ordissolution of the material constituting the carrier, change in thestructure or intermolecular distance of the material constituting thecarrier) or changes in the nature of its surface under the action of apancreatic exocrine function-related factor, whereupon the substancecarried on the carrier is released.

For example, the diagnostic preparation for pancreatic exocrine functionof the present invention comprises a substance carried by a carriercomposed of a material that can be degraded with a pancreatic exocrineenzyme or can be dissolved at pH of the intestinal juice which isincreased due to the secretion of bicarbonate ions from the pancreas.Suppose this diagnostic preparation is orally administered to a subject;if the pancreatic exocrine function of the subject is low, the rate ofdegradation or dissolution of the carrier is also low and the releaserate of the substance carried by the carrier is reduced. The substancereleased from the carrier is absorbed through the intestine, migratesinto the blood, and then excreted either as such or in the form of itsdegradation product or metabolite into the urine or breath. Thediagnosis for a decrease of pancreatic exocrine function can be made bycomparing the amount of the released substance or the degradationproduct or metabolite thereof in the blood, urine or breath atpredetermined time points after the administration or by comparing thetime course of that amount.

The carrier is preferably composed of a material (substrate) that can bedegraded with a pancreatic exocrine enzyme (e.g., protease, lipase,amylase) or a material that can be dissolved at pH of the intestinaljuice (e.g., pH 4.5 to 8.5). The material used as a substrate may beproperly selected depending on the type of the pancreatic exocrineenzyme to be tested.

The material that can be degraded with a protease may be any one thatcontains an amide bond or a peptide bond. Specific examples includeproteins (e.g., egg white, gelatin, poly-L-lysine, albumin), peptides,polypeptides, cross-linked peptides and proteoglycans. The material thatcan be degraded with lipase may be any one that contains an ester bond,such as triglycerides and diglycerides. Specific examples include lipids(e.g., glycerin mono-fatty acid esters, glycerin di-fatty acid esters).Among them, glycerin monocaprylate, glycerin monostearate andpolyethylene glycol monostearate are preferred. The material that can bedegraded with amylase may be any one that contains an α-1,4-glycosidebond. Specific Examples include polysaccharides (e.g., starch, amylose,amylopectin, dextrin) and cross-linked oligosaccharides.

The material that can be dissolved at pH of the intestinal juice may beany compound that is commonly used in enteric preparations. Specificexamples include cellulose acetate phthalate, carboxymethylethylcellulose, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethylcellulose acetate succinate, methacrylate copolymer L, methacrylatecopolymer LD, methacrylate copolymer S and shellac.

The aforementioned materials may be used alone or in combination as amixture of two or more of them. The materials are not limited to thosespecifically mentioned above.

The administered substance may be any compound or element. The type ofthe substance is not limited, as long as the amount in which thesubstance or a degradation product or metabolite thereof migrates intothe blood or is excreted out of the body (e.g., the amount of excretionin the urine or breath) can be determined.

In the case of determining the amount of migration into the blood, theadministered substance or a degradation product or metabolite thereof ispreferably such that it can migrate rapidly into the blood through thelumen of the digestive tract. The administered substance or adegradation product or metabolite thereof is also preferably such thatit can be directly determined in the whole blood, serum or plasma,either as such or after pretreatment (e.g., deconjugation) orisolation/purification procedures, by a colorimetric, fluorometric,electrochemical or radiochemical/nuclear chemical method or the like.Even if the administered substance or a degradation product ormetabolite thereof cannot be determined directly, they may be renderedindirectly quantifiable by converting them, either enzymatically or bymeans of a color reagent, an oxidation-reduction reagent or the like,into a substance that can be determined by a calorimetric, fluorometricor electrochemical method or the like.

Specifically, the substance which, either in the form of theadministered substance or as a degradation product or metabolitethereof, can be directly determined by a colorimetric method includes,but is not limited to, indocyanine green, bromo sulphalein, neutral red,phenolsulfonephthalein and indigo carmine which all have a chromophore.The substance which can be determined directly by a fluorometric methodincludes, but is not limited to, fluorescent substances such asfluorescein, calcein and rhodamine. The substance which can bedetermined directly by an electrochemical method includes, but is notlimited to, oxidation-reduction substances such as ferrocenecarboxylicacid, ascorbic acid, lithium ion and glutathione. The substance whichcan be determined directly by a radiochemical/nuclear chemical methodincludes, but is not limited to, compounds labeled with a radioisotopeor stable isotope. The substance that can be determined indirectly by acolorimetric method includes, but is not limited to, p-aminobenzoicacid, 4-aminoantipyrine, sodium iodide, caffeine, xylose, alcohols(e.g., ethanol), organic acids (e.g., formic acid, acetic acid,propionic acid, pyruvic acid, benzoic acid) and saccharides (e.g.,glucose). The substance that can be determined indirectly by afluorometric method includes, but is not limited to, fluoresceindiacetate. The substance that can be determined indirectly by anelectrochemical method includes, but is not limited to, galactose,D-amino acids and glycolic acid.

In the case of determining the amount of excretion in the urine, theadministered substance or a degradation product or metabolite thereof ispreferably such that it can be excreted rapidly into the urine. Theadministrated substance or a degradation product or metabolite thereofis also preferably such that it can be directly determined in the urine,either as such or after pretreatment (e.g., deconjugation) orisolation/purification procedures, by a colorimetric, fluorometric,electrochemical or radiochemical/nuclear chemical method or the like.Even if the administered substance or a degradation product ormetabolite thereof cannot be determined directly, they may be renderedindirectly quantifiable by converting them, enzymatically or by means ofa color reagent, an oxidation-reduction reagent or the like, into asubstance that can be determined by a colorimetric, fluorometric orelectrochemical method or the like.

Specifically, the substance which, either in the form of theadministered substance or as a degradation product or metabolite thereofcan be directly determined by a calorimetric method includes, but is notlimited to, phenolsulfonephthalein and indigo carmine which all have achromophore. The substance which can be determined directly by afluorometric method includes, but is not limited to, fluorescentsubstances such as fluorescein, calcein and rhodamine. The substancewhich can be determined directly by an electrochemical method includes,but is not limited to, oxidation-reduction substances such asferrocenecarboxylic acid, ascorbic acid and lithium ion. The substancewhich can be determined directly by a radiochemical/nuclear chemicalmethod includes, but is not limited to, compounds labeled with aradioisotope or stable isotope. The substance which can be determinedindirectly by a calorimetric method includes, but is not limited to,p-aminobenzoic acid, 4-aminoantipyrine, sodium iodide, caffeine, xylose,alcohols (e.g., ethanol), organic acids (e.g., formic acid, acetic acid,propionic acid, pyruvic acid and benzoic acid, and saccharides (e.g.,glucose). The substance which can be determined indirectly by afluorometric method includes, but is not limited to, fluoresceindiacetate. The substance which can be determined indirectly by anelectrochemical method includes, but is not limited to, galactose,D-amino acids and glycolic acid.

In the case of determining the amount of excretion in the breath, theadministered substance or a degradation product or metabolite thereof ispreferably such that it is volatile or can be rapidly degraded ormetabolized into CO₂ or ammonia. The administered substance or adegradation product or metabolite thereof is also preferably such thatit can be directly determined in the breath, either as such or afterpretreatment (e.g., trapping, concentration) or isolation/purificationprocedures, by a colorimetric, fluorometric, electrochemical orradiochemical/nuclear chemical method or the like. Even if theadministered substance or a degradation product or metabolite thereofcannot be determined directly, they may be rendered indirectlyquantifiable by converting them, either enzymatically or by means of acolor reagent, an oxidation-reduction reagent or the like, into asubstance that can be determined by a colorimetric, fluorometric orelectrochemical method or the like.

The substance which, either in the form of the administered substance oras a degradation product or metabolite thereof, can be directlydetermined by a radiochemical/nuclear chemical method includes, but isnot limited to, compounds labeled with a radioactive isotope (e.g., ¹⁴C)or a stable isotope (e.g., ¹³C, ¹⁵N). Specifically, the substanceincludes, but is not limited to, organic acids such as ¹³C-labeledNaHCO₃, ¹³C-labeled formic acid, ¹³C-labeled acetic acid, ¹³C-labeledpropionic acid, ¹³C-labeled lactic acid, ¹³C-labeled butyric acid,¹³C-labeled pyruvic acid and ¹³C-labeled benzoic acid; ¹³C-labeled aminoacids such as ¹³C-labeled alanine; ¹³C-labeled saccharides such as¹³C-labeled glucose; and ¹³C-labeled alcohols such as ¹³C-labeledethanol.

The diagnostic preparation for pancreatic exocrine function of thepresent invention is preferably formulated into an oral dosage form(e.g., tablet, capsule, powder, granule, liquid) that can be deliveredto the intestine without being affected in the stomach. Preferably, thediagnostic preparation is in the form of a liquid preparation havingmicrocapsules or microspheres dispersed or emulsified in water. As thepharmaceutical additives for the formulation of the preparation, variousexcipients, aids and additives known in the art can be used. Forexample, the excipient includes saccharides (e.g., lactose, sucrose,glucose, mannitol), starches (e.g., potato starch, wheat starch, cornstarch), inorganic compounds (e.g., calcium carbonate, calcium sulfate,sodium hydrogen carbonate, sodium chloride), crystalline cellulose,plant powder (e.g., licorice powder, gentian powder) and the like. Thebinder includes starch paste, gum arabic, gelatin, sodium alginate,methyl cellulose, ethyl cellulose, polyvinyl pyrrolidone, polyvinylalcohol, hydroxypropyl cellulose, carmellose and the like. Thedisintegrant includes starch, agar, gelatin powder, crystallinecellulose, sodium carboxymethyl cellulose, calcium carboxymethylcellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginateand the like. The lubricant includes magnesium stearate, talc,hydrogenated plant oils, macrogol, silicone oils and the like. Thesuspending agent includes gum arabic, sodium alginate, methyl cellulose,sodium carboxymethyl cellulose, polyvinyl pyrrolidone, polysorbate 80,gum tragacanth, aluminum monostearate and the like. The emulsifyingagent includes gum arabic, cholesterol, polyoxyl 40 stearate, sorbitansesquioleate, polysorbate 80, sodium lauryl sulfate and the like. Inaddition, purified water and surfactants may also be used.

In one embodiment of the dosage form of the diagnostic preparation forpancreatic exocrine function of the present invention, a preparation inwhich a substance to be administered is carried by a polymeric matrix isprovided. As the material forming the polymeric matrix, any material maybe selected such that the structure of the matrix is changed under theaction of a pancreatic exocrine function-related factor so that thesubstance can be released from the matrix. Such material includes, e.g.,those substances which can be degraded with any one of theaforementioned pancreatic exocrine enzymes and those substances whichcan be dissolved at pH of the intestinal juice. The preparation in whichthe substance to be administered is carried by the polymeric matrix maybe further covered with an enteric coating.

In another embodiment, a preparation in which a core including thesubstance to be administered is covered with an outer coating isprovided. As the material that forms the outer coating, any material maybe selected such that the structure of the outer coating is changedunder the action of a pancreatic exocrine function-related factor sothat the substance can be released from the core. Such materialincludes, e.g., those substances which can be degraded with any one ofthe aforementioned pancreatic exocrine enzymes and those substanceswhich can be dissolved at pH of the intestinal juice. Two or more kindsof the material may be used so as to form a laminated outer coating. Thepreparation in which the core including the substance to be administeredis covered with the outer coating may be further covered with an entericcoating. The core may be a tablet, capsule or granule.

In the preparation in which the substance to be administered is carriedby the polymeric matrix or the preparation in which the core includingthe substance to be administered is covered with the outer coating, theparticle diameter is preferably 2 nm to 20 mm, preferably 1 μm to 8 mm,more preferably 1 μm to 500 μm. For the purpose of increasing thegastric emptying rate of the preparation, a substance having highspecific gravity (e.g., gold), an alkali (e.g., sodium hydrogencarbonate) or a component that can enhance gastric emptying (e.g.,metoclopramide) may be added to the preparation, or the surface of thepreparation may be treated to render it hydrophilic.

The preparation in which the substance to be administered is carried bythe polymeric matrix or the preparation in which the core including thesubstance to be administered is covered with the outer coating ispreferably microencapsulated and suspended or emulsified in water. Theprocesses for producing microencapsulated preparations are known in theart and described in, e.g., Patric V. Deasy, Microencapsulation andRelated Drug Process, Marcel Dekker Inc., New York (1984); U.S. Pat. No.4,061,254; U.S. Pat. No. 4,818,542; U.S. Pat. No. 5,019,400; U.S. Pat.No. 5,271,961; and Wakiyama et al., Chem. Pharm. Bull., 29, 3363-68(1981).

Alternatively, the preparation in which the substance to be administeredis carried by the polymeric matrix or the preparation in which the coreincluding the substance to be administered is covered with the outercoating may be filled in a capsule. In this case, it is preferred thatthe capsule itself be enteric, or soluble in the intestine.

The amount in which the substance to be administered is contained in thepreparation may vary depending on the type of the preparation.Generally, the content is 1 to 99% by weight, preferably 1 to 10% byweight.

The preparation described above can also be used as a delivery systemfor a medical agent that targets the superior part of the smallintestine, or as an enteric preparation.

In the pancreatic exocrine function test using the diagnosticpreparation for pancreatic exocrine function of the present invention,the diagnostic preparation for pancreatic exocrine function of thepresent invention is orally administered. The pancreatic exocrinefunction is then diagnosed either from the data of the integral of theamounts in which the administered substance or a degradation product ormetabolite thereof migrated into the blood or were excreted out of thebody (e.g., the excreted amount in the urine or breath) at predeterminedtime points after the administration or from the data of the time course(onset slope, change in slope, peak time, etc.) of those amounts duringa predetermined time period after the administration.

The method for the measurement of the amount of the administeredsubstance or the degradation product or metabolite thereof is properlyselected depending on the nature or type of the material to be tested(e.g., blood, urine, breath) and the substance to be administered.Examples of the method include colorimetry, fluorometry, massspectrometry, NMR (nuclear magnetic resonance), HPLC, gaschromatography, gas chromatography-mass spectrometry (GC-MS),photoelectric acoustic spectroscopy, GM counter method, liquidscintillation, solid scintillation, autoradiography, and ionizationchamber method.

Specifically, for determining the amount of migration into the blood,the collected blood may be measured directly, or the blood that ispreliminarily subjected to some treatment (e.g., isolation,pretreatment) may be measured. For example, determination can be carriedout by administering a diagnostic preparation containing a fluorescentcompound to a subject, collecting the blood from the subject atpredetermined time points after the administration, preparing serum orplasma from the blood, and then comparing the fluorescence intensity inthe serum or plasma. In the case where a diagnostic preparationcontaining a pigment is administered, the blood is collected from thesubject at predetermined time points after the administration and theconcentration of the pigment in serum or plasma is then determined witha spectrophotometer or the like. Alternatively, the amount of thepigment in the blood may be determined externally on the skin of thesubject without collecting the blood.

In the case where the urine is used as a sample, determination can bemade by colorimetry, fluorometry, mass spectrometry, NMR (nuclearmagnetic resonance), HPLC, gas chromatography or the like. The urine maybe measured directly, or the urine that is preliminarily subjected tosome treatment (e.g., isolation, pretreatment) may be measured. Forexample, determination can be carried out by administering a diagnosticpreparation containing a fluorescent compound to a subject, collectingthe urine from the subject at predetermined time points after theadministration, and then comparing the fluorescence intensity in theurine. In the case where a diagnostic preparation containing a pigmentis administered, the urine is collected from the subject atpredetermined time points after the administration and the concentrationof the pigment in the urine is then determined with a spectrophotometeror the like.

For determining the amount of excretion in the breath using, e.g., adiagnostic preparation containing a ¹³C-labeled compound, determinationcan be carried out by gas chromatography-mass spectrometry (GC-MS),infrared spectroscopy, mass spectroscopy, photoelectric acousticspectroscopy, NMR (nuclear magnetic resonance) or the like on ¹³CO₂. Inthe case where a diagnostic preparation containing a ¹⁴C-labeledcompound is used, the breath, either directly or after trapping CO₂ in asolvent, can be subjected to measurement with a GM counter, a liquidscintillation counter or a solid scintillation counter or byautoradiography, ionization chamber method or the like.

Prior to the administration of the diagnostic preparation for pancreaticexocrine function of the present invention to a subject, a test meal orthe like may be given to the subject to stimulate the secretion ofpancreatic exocrine enzymes. Alternatively, the diagnostic preparationmay be administered together with a test meal.

The substance to be administered must be contained in the diagnosticpreparation for pancreatic exocrine function of the present invention insufficient amount to enable determining or confirming the increase ofthe substance to be detected (e.g., the substance administered or adegradation product or metabolite thereof) in the sample afteradministration. It will be varied depending on the age and body weightof a subject and the intended purpose of the test. For example, the unitdose may be about 1 to 2,000 mg/kg of body weight for an adult.

The drug delivery system and the enteric preparation provided by thepresent invention enables delivering a medical agent in such a way thatit can be absorbed through the digestive tract including and downstreamof the duodenum. Accordingly, they are useful for the delivery of amedical agent which presents inconvenience if degraded with gastric acidor an enzyme in the stomach or which may cause a disorder if absorbedthrough the stomach. Examples of the medical agent include iron agents(e.g., ferric pyrophosphate, soluble), bifidus preparations, laxativesfor constipation (e.g., bisacodyl, sennoside) and aspirin.

Since the drug delivery system and enteric preparation provided by thepresent invention have a property of delivering a medical agent in sucha way that it can be absorbed through the digestive tract including anddownstream of the duodenum, they can provide controlled release of amedical agent. Therefore, by utilizing its delayed release, the medicalagent can be prevented from causing any adverse side effects whilesustaining its beneficial effects. Examples of the medical agent includecephem antibiotics (e.g., pindolol, cephalexin), chlorpheniraminemaleate, griseofulvin and etretinate.

The present application claims priority to Japanese Patent ApplicationNo. 2000-272116, the disclosure and/or the drawings of which areincorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course plot of the amount of trypan blue(absorbance at 590 nm) eluted from trypan blue-containing solidified eggwhite.

FIG. 2 shows the time course plot of the amount of trypan blue(absorbance at 590 nm) eluted from trypan blue-containing starch gel.

FIG. 3 shows the time course plot of the degree of increase of Δ¹³C(‰)starting five minutes after the administration of ¹³C—NaHCO₃— containingsolidified white egg onward.

FIG. 4 shows the time course plot of Δ¹³C(‰) after the administration of[1-¹³C]alanine-containing starch gel.

FIG. 5 shows the time course plot of the amount of ethanol (absorbanceat 560 nm) released from albumin-ethyl ester fine particles.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention is illustrated in more detail by thefollowing examples, however the scope of the present invention shall notbe limited by the examples.

EXAMPLE 1

Solidified egg white containing trypan blue was immersed in a bufferwith or without trypsin, and the time course of trypan blue elution wascompared.

1-1: Method

A small amount of trypan blue was added to egg white and dissolvedtherein by fully stirring while preventing the formation of foam. Theegg white colored in dark blue was put in a dialyzing tube (diameter: 5mm, MWCO: 12-14,000), and boiled in a 0.4% trypan blue solution until itsolidified. After 7 or 8 minutes, the dialyzing membrane was cut andremoved to give two pieces of cylinder-shaped solidified egg white (5 mmin diameter×25 mm in length).

One piece of the solidified egg white was immersed in (i) 15 mL of 200mM Pipes-K (pH 7.0), and the other was immersed in (ii) 15 mL of 200 mMPipes-K (pH 7.0) containing 0.1% trypsin.

An aliquot (200 μl) was sampled from each of the solutions over time,and the amount of eluted trypan blue was determined by measuring theabsorbance at 590 nm with a microplate reader (Molecular Device).

1-2: Results

At 40 minutes after the immersion was initiated, the absorbance at 590nm was 0.056 in (i) 5 mL of 50 mM Tris-HCl (pH 8.0) and 0.099 in (ii)the buffer with trypsin. Thus, the elution of trypan blue was enhancedover the blank by the addition of trypsin (FIG. 1). At 165 minutes afterthe immersion was initiated, the absorbance at 590 nm was 0.092 in (i) 5mL of 50 mM Tris-HCl (pH 8.0) and 0.338 in (ii) the same buffer withtrypsin. Thus, a significant difference was observed.

EXAMPLE 2

A starch gel containing trypan blue was immersed in each of a bufferwith the duodenal juice, a buffer with amylase and a buffer, and thetime course of trypan blue elution was compared.

2-1: Method

A hydrolyzed starch for electrophoresis (Wako) was added to a 0.16%trypan blue solution (0.35% NaCl) to give a final concentration of 10%and then heated while stirring. When the mixed solution was viscous andclear, the solution was aspirated with a disposable measuring pipette(diameter: 4 mm) while preventing the formation of foam and solidifiedin cold water. After cooling for 1 hour, the gelated starch was pushedout of the measuring pipette, thereby giving three cylinder-shapedstarch gels (4 mm in diameter×10 mm in length).

The gels were immersed in (i) 5 mL of 50 mM Tris-HCl (pH 8.0), (ii) 5 mLof 50 mM Tris-HCl (pH 8.0) plus 1 mL of a 344 U/L amylase solution and(iii) 5 mL of 50 mM Tris-HCl (pH 8.0) plus 1 mL of the duodenal juice(the duodenal extract solution), respectively.

An aliquot (200 μl) was sampled from each of the solutions over time,and the amount of eluted trypan blue was monitored by measuring theabsorbance at 590 nm with a microplate reader (Molecular Device).

The duodenal juice was prepared by removing the duodenum from an 8-weekold male Wistar rat fasted overnight, incising the duodenum, adding theincised duodenum to 1 mL of 50 mM Tris-HCl (pH 8.0), agitating themixture with a vortex mixer and then removing tissues.

2-2: Results

At 30 minutes after the immersion was initiated, the absorbance at 590nm was 0.165 in (i) 5 mL of 50 mM Tris-HCl (pH 8.0), 0.198 in (ii) thebuffer with amylase, and 0.346 in (iii) the buffer with the duodenaljuice. Thus, the elution of trypan blue was enhanced over the blank bythe addition of amylase and the duodenal juice (FIG. 2).

EXAMPLE 3

Solidified egg white containing ¹³C—NaHCO₃ and fluorescein wasadministered intraduodenally to a rat with chronic pancreatitis and acontrol rat under anesthesia. The time course of the ¹³CO₂ concentrationin exhaled CO₂ and the fluorescein concentrations in the blood and urinewere compared.

3-1: Method

40 mg of ¹³C—NaHCO₃ (Mastorace) and 3.2 mg of fluorescein were dissolvedin 1 mL of distilled water, and then 7 mL of egg white was added. Theresulting solution was mixed gently by pipetting while preventing theformation of foam. The mixed solution was poured into a chamber slide(Nunc, Cat# 177453), heated at 80° C. for 15 minutes to solidify the eggwhite containing ¹³C—NaHCO₃ and fluorescein. After being left to cool,the solidified egg white was cut out with a disposable measuring pipette(4 mm in diameter), thereby forming a cylinder of solidified egg white(200 μL in volume).

The chronic pancreatitis rat was prepared by injecting oleic acid intothe pancreatic duct of a 5-week old male Wistar rat and then keeping therat for 3 weeks in accordance with the method of Mundlos et al. (Mundloset al., Pancreas 1:29 (1986)). As a control, a rat that had beensubjected to only midline incision was used.

After fasting overnights the chronic pancreatitis rat and the controlrat, both 8 week-old, were fixed supinely under anesthesia. The breathwas collected at a rate of about 100 to 300 ml/min using a stroke pump(Variable Stroke Pump VS-500, Shibata Kagaku Kogyo) and introduceddirectly to the flow cell of a ¹³CO₂ analyzer EX-130S (Nihon Bunko). APerma Pure drier (MD-050-12P, Perma Pure INC.) was placed between therat holder and the stroke pump to remove water vapor from the breath.When the CO₂ concentration stabilized, 200 μL of the solidified eggwhite containing ¹³C—NaHCO₃ and fluorescein was administeredintraduodenally to the rat. The CO₂ concentration in the collectedbreath was held at 3±0.5%.

Output data from the ¹³CO₂ analyzer were AD converted and input to apersonal computer (Apple Power Macintosh 8500). Using a data processingsoftware package Lab VIEW (National Instruments), 10 pieces of data wereintegrated and averaged every 100 msec at intervals of 5 seconds andconverted to ¹³C atom %, Δ¹³C (‰) and CO₂ concentration (%). In thismanner, the ¹³C-breath test was continuously carried out. The converteddata were displayed real time and stored in a hard disc.

Δ¹³C (‰) was calculated from the ¹³C concentration in the exhaled CO₂ ateach time point (¹³C_(tmin)) and the ¹³C concentration in standard CO₂(¹³C_(std)) according to the following equation:Δ¹³C (‰)=[(¹³C_(tmin)−¹³C_(0min))/¹³C_(std)]×1000

After performing the breath test for 20 minutes, the blood and urinewere collected. To the collected blood was added a 3.8% sodium citratesolution at a volume one-sixth of the blood, and the resulting solutionwas centrifuged at 10,000 g for 20 minutes to give a plasma as thesupernatant.

The amount of fluorescein in the plasma was determined following thedescription in the Journal of Osaka Medical University 52(2):27-35. 50μL of the plasma was added with 400 μL of a 0.5N KOH solution inethanol, and the mixed solution was heated at 70° C. for 1 hour and thencooled. To the resulting solution was added 800 μL of 0.15M MgSO₄, andthe solution was centrifuged at 10,000 g for 5 minutes. The amount offluorescein in the supernatant was determined with a fluorescencemicroplate reader (Molecular Device) (excitation wavelength: 485 nm,fluorescence wavelength: 538 nm, sample load amount: 200 μL/well). Theamount of fluorescein in an untreated plasma was also determined with afluorescence microplate reader (Molecular Device).

The amount of fluorescein in the urine was determined following themethod described in Archives of Disease in Childhood (1986) 61:573. 50μL of the urine was added with 450 μL of 0.1N NaOH, and the resultingsolution was heated at 70° C. for 10 minutes. The amount of fluoresceinwas measured with a fluorescence microplate reader (Molecular Device)(excitation wavelength: 485 nm, fluorescence wavelength: 538 nm, sampleload amount: 200 μL/well).

3-2: Results

The degree of increase of Δ¹³C (‰) starting 5 minutes after theadministration onward was calculated according to the followingequation:Degree of increase of Δ¹³C (‰)=Δ¹³C (‰)_(tmin)−Δ¹³C (‰)_(5min)

The comparison of the time course revealed that the degree of increaseof Δ¹³C (‰) starting 5 minutes after the administration onward in thechronic pancreatitis rat was smaller than that in the control rat (FIG.3). The degree of increase of Δ¹³C (‰) at 10 minutes after theadministration was 9.1‰ in the control rats (n=3) and decreased to 3.9‰in the chronic pancreatitis rats (n=3). The degree of increase of Δ¹³C(‰) at 20 minutes after the administration was −9.5‰ in the control ratsand decreased to −13‰ in the chronic pancreatitis rats.

The fluorescein concentration (fluorescence intensity) in thedeconjugated plasma at 20 minutes after the administration was 2.16 RFUin the chronic pancreatitis rats (n=3), which was lower than the valueof 2.69 RFU in the control rats (n=3). The fluorescein concentration(fluorescence intensity) in the untreated plasma was 20.18 RFU in thechronic pancreatitis rats (n=3), which was lower than the value of 29.43RFU in the control rats (n=3). The fluorescein concentration(fluorescence intensity) in the urine at 20 minutes after theadministration was 1.53 RFU in the chronic pancreatitis rats (n=3),which was again lower than the value of 4.05 RFU in the control rats(n=3).

The results demonstrate that the diagnosis of the decrease in pancreaticexocrine function can be achieved by administering ¹³C—NaHCO₃ andfluorescein contained in an egg white carrier.

EXAMPLE 4

A starch gel containing [1-¹³C]alanine and fluorescein was administeredintraduodenally to chronic pancreatitis rats and control rats underanesthesia. The time course of the ¹³CO₂ concentration in exhaled CO₂and the concentrations of fluorescein in the blood and urine after theadministration were compared.

4-1: Method

0.5 g of a hydrolyzed starch for electrophoresis (Wako), 40 mg of[1-¹³C]alanine (Masstrace) and 3 mg of fluorescein were added to 5 mL of50 mM borate buffer (pH 8.5), and the solution was stirred while heatingby boiling in hot water. When the solution became viscous and clear,about 300 μL of the solution was aspirated with an insulin injectingsyringe with a cut tip (Terumo, for 0.5 mL injection) while preventingthe formation of foam, and then left to cool to cause gelation. Thesyringe was cut so that the volume of the gelated starch contained inthe syringe was 150 μL.

Thereafter, the same procedure as in Example 3 was performed.

4-2: Results

The values of Δ¹³C (‰) in the chronic pancreatitis rats were smallerthan those in the control rats (FIG. 4). Δ¹³C (‰) at 5 minutes after theadministration was 42.2‰ in the control rats (n=3) and decreased to32.2‰ in the chronic pancreatitis rats (n=2).

The fluorescein concentration (fluorescence intensity) in the plasma at20 minutes after the administration was 6.67 RFU in the chronicpancreatitis rat (n=1), which was lower than the value of 9.33 RFU inthe control rats (n=3). The fluorescein concentration (fluorescenceintensity) in the urine at 20 minutes after the administration was 4.14RFU in the chronic pancreatitis rats (n=2), which was obviously lowerthan the value of 60.50 RFU in the control rats (n=2).

The results demonstrate that the diagnosis of the decrease in pancreaticexocrine function can be achieved by administering [1-¹³C]alanine andfluorescein contained in a starch gel carrier.

EXAMPLE 5

Fine particles of albumin-ethyl ester were prepared, added to theduodenal juice from a normal rat and a chronic pancreatitis rat, and thetime course of the concentration of ethanol released from the fineparticles was compared.

5-1: Method

26 mL of thionyl chloride (Wako) was slowly added dropwise to 100 mL ofethanol (Wako) that had been cooled to 0° C. in an argon atmosphere. Thesolution was stirred for 10 minutes and then warmed to room temperature.To the solution now at room temperature, 6.64 g of human serum albuminwas added and the mixture was stirred for 24 hours. The reactionsolution was filtered, and the residue was dried with nitrogen gas,thereby giving 7.6453 g of albumin-ethyl ester.

Fine particles of the albumin-ethyl ester were prepared following themethod of Gallo et al. (1984) (International Journal of PharmaceuticsVol. 22 (1984) pp. 63-74). 0.5 mL of 13.3% albumin-ethyl ester was addedto 30 mL of olive oil, and the resulting solution was subjected tosonication (125 W) for 2 minutes while cooling in ice. The solution wastransferred to a 50 mL syringe and added dropwise to 100 mL of olive oilthat had been heated to 125° C. at a rate of 1 to 2 drops/sec. Afterstirring at 1,500 rpm for 10 minutes, the solution was allowed to standfor 1.5 hours and then cooled to room temperature. After adding 60 mL ofether and stirring, the solution was centrifuged at 3,000 g for 15minutes. The resulting precipitate was added with 25 mL of ether,suspended by sonication, and then centrifuged at 3,000 g for 15 minutes.After repeating the same procedure once again, the resulting precipitatewas resuspended in 10 mL of ether and allowed to stand in a draft atroom temperature to remove the ether.

To 25 μL of the resulting suspension of albumin-ethyl ester fineparticles (10 mg/mL in D.W.) were added 50 μL of a solution of 150 U/mLof alcohol dehydrogenase (Sigma) in 360 mM phosphate buffer (pH 7.8),100 μL of a color reagent (38 mM NAD, 1.0 mM nitrotetrazolium blue, 0.1mM 1-methoxy PMS, 88 mM sodium acetate) and 5 μL of the duodenal juice.The solution was allowed to react in a microplate at 37° C. As a blank,a reaction was performed by adding distilled water in place of theduodenal juice. The amount of released ethanol was monitored bymeasuring the absorbance at 560 nm over time with a microplate reader(Molecular Device).

The duodenal juice was prepared by removing the duodenum from each of anormal rat and a chronic pancreatitis rat, incising the duodenum,immersing the incised duodenum in 2 mL of 50 mM carbonate buffer, andthen centrifuging the solution at 10,000 g for 15 minutes.

5-2: Results

At 2 minutes after the duodenal juice was added, the release of ethanolinto the sample containing the duodenal juice from the normal rat wasenhanced compared with the sample containing the duodenal juice from thechronic pancreatitis rat (FIG. 5). At 10 minutes after the duodenaljuice was added, the absorbance at 560 nm was 0.156 in the sample withthe duodenal juice from the normal rat and 0.086 in the sample with theduodenal juice from the chronic pancreatitis rat. Thus, a difference wasobserved.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference.

APPLICABILITY OF THE INVENTION

The preparation for the diagnosis of pancreatic exocrine function of thepresent invention allows a simple pancreatic exocrine function test thatimparts low stress on a subject, is less costly and gives accurateresults in a short time. The test method can be used in pancreatitisscreening in mass physical examination or total health check,determination of the severity of chronic pancreatitis, prediction of thepossibility toward aggravation of fulminating pancreatitis of whichmortality is still high (30%), diagnosis of the etiology ofpancreatitis, and early diagnosis of pancreatic cancer. The test methodis also useful as a diagnostic method for denial of the possibility ofpancreatitis in the diagnosis of general outpatients.

The preparation can also be used as a drug delivery system which targetsthe superior part of the small intestine and as an enteric preparation.

1. A method for ascertaining a level of pancreatic exocrine function,comprising: administering to a subject albumin attached to a substancevia an ester bond, wherein albumin is subject to degradation by apancreatic exocrine enzyme to release the substance from albumin;wherein the substance is an alcohol, organic acid, saccharide or aminoacid; and determining an amount of the substance administered to thesubject or a degradation product or metabolite of the substance thatmigrated into the blood and/or is excreted out of the body of thesubject, to ascertain a level of the pancreatic exocrine function of thesubject.
 2. The method of claim 1, wherein the substance is ¹³C- or¹⁴C-labeled such that the amount of the substance administered to thesubject or a degradation product or metabolite product of the substance,which migrated into the blood and/or excreted out of the body of thesubject, is determined by a radiochemical/nuclear chemical method. 3.The method of claim 1, wherein the amount of the substance or adegradation product or metabolite of the substance is determined fromthe blood, urine, or breath of the subject.
 4. The method of claim 1,wherein the substance is ethanol.
 5. The method of claim 2, wherein thesubstance is ¹³C-labeled.
 6. The method of claim 5, wherein the albuminis unlabeled.